Oral cancer is common among men in the developed world and among the most difficult neoplasms to treat. The growth and metastasis of all solid tumors requires induction of angiogenesis, the creation and remodeling of new blood vessels, to meet the increasing metabolic demands of rapidly dividing transformed cells, thus making the development of anti-angiogenic agents an appealing treatment strategy. Recent studies have identified increased expression of hypoxia- inducible factor (HIF)-1, a transcription factor that promotes angiogenesis, in many different primary and metastatic tumors, suggesting that its activation is common in human cancer (1). We now know that proteins involved in transmitting axonal guidance cues can also play a role in tumor-induced angiogenesis. For example, we were the first to show that Plexin-B1, a protein previously identified as a regulator of neuron growth cone progression and migration and nerve bundle fasciculation, is also highly expressed in endothelial cells and promotes a pro-angiogenic response when bound by its ligand Semaphorin 4D (Sema4D) (3), a protein expressed in head and neck squamous cell carcinomas (HNSCC) and many other solid tumors that enhances their growth and vascularity (4). The broad, long-term objectives of this application are to elucidate the mechanisms of regulation of Sema4D and the implications for tumor- induced angiogenesis. The hypothesis to be tested is that Sema4D is upregulated in hypoxia due to HIF-1- mediated pathways, and that its expression acts with other HIF-1-regulated gene products such as membrane type 1-matrix metalloproteinase (MT1-MMP) and vascular endothelial growth factor (VEGF) to induce a more vascular and consequently a more aggressive tumor phenotype. The specific aims are: 1) to establish the mechanism of transcriptional regulation of Sema4D and its biological relevance in hypoxia- mediated tumorigenesis. This will be accomplished by analyzing Sema4D protein and message levels in normoxic and hypoxic cells expressing reduced or constitutively active HIF, thorough promoter analysis of the Sema4D gene and through in vitro and in vivo angiogenesis assays and tumor xenograft experiments; 2) to determine the importance of hypoxia-mediated induction of MT1-MMP on the ability of Sema4D to induce angiogenesis. This will be studied through analysis of MT1-MMP levels in cells with altered HIF activity and the biological significance of MT1-mediated processing of Sema4D in a tumor xenograft model, and; 3) to determine VEGF and Sema4D contributions to HNSCC-induced angiogenesis through retroviral-mediated gene transfer directly into tumor cells in a conditional knockout mouse model. I believe that these investigations into Sema4D/Plexin-B1-mediated angiogenesis will support a newly emerging model of tumor-induced angiogenesis and present possible new targets for cancer therapy. PUBLIC HEALTH RELEVANCE: By their very nature, tumor cells are genetically unstable, a trait that contributes to their ability to proliferate inappropriately, avoid natural defenses and acquire resistance to chemotherapy. The development of anti-angiogenic agents capable of blocking a tumor's blood supply has become an intriguing way to circumvent this problem. If successful, our proposed project may uncover an important mechanism for tumor-induced angiogenesis, thereby presenting new targets for the development of anti- angiogenic therapies for the management of aggressive tumors.